LsNAC72 from a major QTL activates LsNRAMP7 expression to promote iron accumulation in lettuce

Iron (Fe) is an essential micronutrient critical for plant growth and development, as well as for human health. Lettuce (Lactuca sativa) serves as a rich source of Fe for consumers. However, little is known about the genetics underlying Fe concentration in lettuce. In this study, we performed a genome-wide association study (GWAS) of Fe concentration using the multiparent advanced generation intercross population derived from 16 founder lines. A significant locus associated with Fe concentration was identified on chromosome 7. Sequence and expression analysis suggested that the gene LsNAC72, which encodes a NAC transcription factor within this significant region, is the causal gene associated with variations in Fe concentration. Knockout of the LsNAC72 gene significantly decreased Fe concentration in the leaves of the Lsnac72 mutants under Fe-deficient conditions, leading to reduced growth. Furthermore, overexpression of LsNAC72 enhanced Fe accumulation in leaves and roots under both Fe-sufficient and Fe-deficient conditions. Further analysis confirmed that LsNAC72 directly binds to the promoter and activates the expression of LsNRAMP7, which encodes an Fe transporter. The roots and leaves of the Lsnramp7 knockout mutants had significantly lower Fe concentrations than those of the wild-type under both Fe-sufficient and Fe-deficient conditions. The identification of the transcription factor LsNAC72 and NRAMP homologs in this study sheds light on the regulation of Fe concentration in plants, and the identification of natural variations in LsNAC72 alleles provides a genetic resource for lettuce breeding programs.